Estradiol

Thymi were dissected into ice-cold PBS, and adipose and connective tissue were removed, transferred into about 5 ml of steroid-free culture medium [RPMI 1640 supplemented with 10% charcoalstripped heat-inactivated FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 25 mM Hepes, 0.1 mM nonessential amino acids, and gentamicin (50 mg/ml)], and minced into 1-mm³ pieces using forceps and scalpel. Fragments were left for at least 10 min to deplete endogenous steroid substrates and then transferred with forceps to 24-well plates containing 400 ml of steroid-free culture medium. Samples were cultured for 72 hours at 37°C in 5% CO₂, and then supernatants were collected and immediately assayed or stored at −20°C until assayed for steroids. Steroids were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (corticosterone, Arbor Assays, K014; progesterone, Cayman Chemical, 582601; testosterone, Cayman Chemical, 582701; estradiol, Cayman Chemical, 501890) according to the manufacturer’s protocol, with standards diluted in steroid-free culture medium. One outlier subject, with all steroid measurements greater than threefold higher than all other subjects, was omitted from these analyses. Inclusion of this subject did not alter the results of these experiments.